The mouse ovary serves as a paradigm for investigating the developmental biology of mammalian gonadogenesis, oogenesis and fertilization. Gonadogenesis: Mouse gestation occurs over 20 days. Germ cells, first detected in the developing embryo 7.5 days post coitus (dpc), migrate from the allantois to the genital ridge by 12.5 dpc. In XX females, the primitive gonad then differentiates into an ovary. Sex-specific cDNA libraries have been prepared from female and male genital ridges isolated 12-13 dpc. A subtractive cloning strategy is being used to identify female-specific gene products involved in early organogenesis of the ovary. Oogenesis: The expression of the zona pellucida, an ovary-specific extracellular matrix composed of three glycoproteins (ZP1, ZP2, ZP3), serves as a marker of oocyte growth and differentiation in the adult female. The mouse and human ZP2 and ZP3 genes, transcripts and proteins have been characterized. A 12bp DNA sequence 200 bp upstream of the start of transcription of all four genes is necessary and sufficient for zona promoter activation of reporter genes microinjected into growing mouse oocytes. Efforts are currently underway to clone ZAP-1, the putative transcription factor that binds to this DNA element. These studies will provide important molecular details of mechanisms involved in the coordinate, oocyte-specific expression of the zona genes. ZAP-1 will additionally provide an early marker of oocyte growth and differentiation. Fertilization: The three zona proteins are secreted and form an extracellular matrix that mediates the relatively species-specific events of fertilization. Using microinjection techniques to degrade endogenous zona transcripts and to introduce synthetic zone mRNAs, we are assessing protein-protein interactions in the assemblage of the zona matrix. To investigate the molecular biology of sperm-egg interactions mediated by the zona, we have established a mouse in vitro fertilization assay. We are using recombinant DNA techniques to create mutant zona proteins and mouse/human chimeric zona proteins. These will be tested for their ability to competitively inhibit fertilization, enabling establishment of structure-function correlations.